Detection of nucleic acids in a sample is useful in diagnostic, therapeutic, forensic, agricultural, food science applications and other areas. One technique for purifying a target polynucleotide, which is often used in diagnostic procedures, involves capturing a target polynucleotide onto a solid support. The solid support retains the target polynucleotide during one or more washing steps of the target polynucleotide purification procedure. The captured target sequence can be analyzed by various methods. One such method uses nucleic acid probes that hybridize to a target sequence. Probes can be designed to detect different target sequences such as those characteristic of microorganisms, viruses, human genes, plant or animal genes, and/or pathogenic conditions. Additional analysis techniques that benefit from captured target nucleic acids include, amplification assays, microarrays, sequencing assays, mass spectrometry of nucleic acids, and other techniques known in the art.
A target nucleic acid can be captured using a mediator or capture polynucleotide that hybridizes to bind both to a target nucleic acid and to a nucleic acid fixed to a solid support. The mediator polynucleotide joins the target nucleic acid to the solid support to produce a complex comprising a bound target nucleic acid. A labeled probe can be hybridized to the bound target and unbound labeled probe can be washed away from the solid support (see Stabinsky, U.S. Pat. No. 4,751,177).
Because hybridization proceeds more rapidly between nucleic acids that are both in solution compared to between one nucleic acid in solution and one immobilized nucleic acid, it is preferable in capturing a target nucleic acid using a capture probe that the capture probe hybridizes to the target nucleic before the capture probe is immobilized by binding to a support-bound polynucleotide. Such can be accomplished by performing the capture under first hybridization conditions in which the capture probe hybridizes to the target nucleic acid to for a target capture probe:target nucleic acid complex, and second hybridization conditions of reduced stringency in which the capture probe of the formed complex binds to an immobilized probe to form a further complex comprising immobilized probe:target capture probe:target nucleic acid (see Weisburg et al, U.S. Pat. No. 6,110,678). This type of assay is facilitated by designing the capture probe and immobilized probe to contain complementary homopolymer sequences of adenine and thymine. The melting temperature of hybrids formed between adenine and thymine is usually less than that formed between complementary sequences that include guanine and cytosine residues.